Viruses are packets of genetic material associated with a few virus-specific proteins. They enter selected cells via specific receptors, replicate within these, using the normal cellular machinery and exit most often by destroying their former hosts. Antiviral strategies have employed immunological techniques or drugs inhibiting virus-specific functions. This has been difficult because agents against many viruses also interfere with normal cellular functions. Because viruses have evolved towards a minimal number of virus-specific functions, appropriating normal, cellular functions instead, virus-specific targets are few in number. Since there are a great variety of viruses, an agent targeted to an activity specific to a given virus is unlikely to act equivalently on a different virus. Because the virus genome mutates frequently, viruses commonly develop resistance against specific, previously effective agents, allowing escaping the selective pressures of chemotherapeutic agents. Thus, of the thousands of antivirals tested, only about 40 continue efficacious, of which one half is anti-HIV agents. Combinations of anti-HIV agents are commonly necessary to achieve significant benefit. Similarly, “antigenic shift” mutations occur often after a vaccine has been employed, making the vaccine less protective (a year or so in the case of influenza) and this is a major problem in strategies against a possible influenza pandemic.
Viruses can be grouped into non-enveloped and enveloped viruses. Enveloped viruses are enclosed within a lipoprotein membrane, or envelope. This envelope is derived from the host cell as the virus “buds” from its surface and consists mostly of lipids not encoded by the viral genome. Even though it carries molecular determinants for attachment and entry into target cells, and is essential for the infectivity of enveloped viruses, it is not subject to drug resistance or antigenic shift.
Although virus envelope lipids derive from the host cell plasma membrane, they are deposited in the envelopes at proportions differing from that membrane. For example, the envelope of HIV is enriched in cholesterol (2.5 times) and in sphingomyelin (3 times), both located mainly in the external lamella of the envelope. (Aloia, et al 1993.) The membranes of influenza viruses are similarly enriched (Scheiffele, et al 1999) and the same pattern has been reported for other enveloped viruses. Importantly, it has recently been shown that cholesterol depletion interferes with the infectivity of enveloped viruses (Ono and Freed, 2001; Simons and Ehehalt, 2002). Indeed, the evidence indicates that the envelopes of many enveloped viruses contain phase separated “lipid rafts” enriched in cholesterol thus suggesting that viral envelope lipids may be a target in the arsenal against enveloped viruses.
Since the raft lipids of virus infected cells are synthesized by these cells, use of cell— directed inhibitors, such as the “statins” will exert too much systemic toxicity to be acceptable as “anti-raft agents”. Indeed anti-raft strategies will be effective only against extra cellular forms of the virus, when these forms are externally accessible, namely in the naso—and oropharynx and respiratory tract (e.g. influenza), the urogenital tract (e.g. HIV), the skin (e.g. herpes simplex) or deposited on surfaces (fomites).
The fact that cholesterol and other lipids can exchange between the phospholipid lamellae of cellular membranes, as well as liposomes, provides important information. McLean and Phillips (1981) point out that the short “half-time”, T1/2, 2-3 min, of cholesterol transfer between liposomes indicates collisions between these particles. Steck et al (2002) support this conclusion. They have shown that all the cholesterol transfer from red cells to an acceptor occurs with a T1/2˜1 sec, depending only of the concentration of the acceptor. They propose an “activation-collision” mechanism, where cholesterol is captured by collision. The T1/2 for the transfer of a fluorescent analogue of sphingomyelin between membranes is ˜21 sec (Bai and Pagano, 1997) and the “off-rate” T1/2 for the transfer of C18 fatty acids from oil to water is ˜1.3 sec (Small, 2002). In contrast, the T1/2 for the transfer of phosphatidyl-choline between liposomes was measured to be ˜48 h at 37° C. (McLean and Phillips, 1981).
These data suggest the possibility that enveloped viruses might be inactivated by exposure to phospholipid liposomes. However, phospholipid liposomes are extremely costly, unstable and are unlikely to be available in the quantities needed for prophylaxes. Moreover phospholipid liposomes cannot readily be made with the low cholesterol content required to give net extraction (rather than the two-way exchange) of this lipid and their production requires the use of organic solvents that are a major source of cellular toxicity.
Liposomes can be used to transport drugs for the delivery of pharmaceutical or cosmetic compositions. For example, International Patent Application WO96/12472 (Chinoin Gyógyszer És Vegyészeti Termékek Gyára R T et al.) disclosed a liposomic composition containing, as active ingredient, (−)-N-alpha-dimethyl-N-(2-propynylphenylethylamine) (selegilin) and/or salt thereof. The disclosed composition contains 0.1-40% by weight of selegilin and/or a salt thereof, 2 to 40% by weight of lipids, preferably phospholipids, 0 to 10% by weight of cholesterol, 0 to 20% by weight of an alcohol, 0 to 25% by weight of a glycol, 0 to 3% by weight of an antioxidant, 0 to 3% by weight of a preserving agent, 0 to 2% by weight of a viscosity influencing agent, 0 to 50% by weight of cyclodextrin or a cyclodextrin derivative and 30 to 90% by weight of water. This application also provides the administration of said composition for the treatment of Alzheimer's disease, Parkinson's disease, depression, stroke, motion sickness or myelitis.
It is also known from WO2005030170 (Université Pasteur et al.) a method for initiating the controlled rupture of the membrane of a biocompatible phospholipid liposome, often called a furtive liposome, thereby releasing the liposome content to the environment thereof. A releasing agent, preferably an α-cyclodextrin, is embodied in the form of a biocompatible molecule.
For the reasons described above, the Applicants have explored the advantages of using liposomes such as non phospholipid Lipid Vesicle (nPLV) composed of single-chain poly-(ethylene glycol)-alkyl ethers [(PEG)-alkyl ethers] instead of phospholipid liposomes (Wallach, 1996; Varanelli et al. 1996; Wallach and Varanelli, 1997).
U.S. Pat. No. 5,561,062 (Varanelli et al.) already provides an in vitro method of inactivating enveloped viruses by using paucilamellar lipid vesicles, preferably having non-phospholipids, and preparations useful in accomplishing this inactivation. The method is based on the discovery that paucilamellar lipid vesicles, preferably having non-phospholipids as their primary structural material, can fuse with enveloped virus and that the nucleic acid of the virus denatures shortly after the fusion. Generally, the paucilamellar lipid vesicle is filled with either an oil solution or a water solution, both containing a nucleic acid degrading agent.
An other patent application, EP 1 304 103 A1 (D. F. H Wallach) provides lipid vesicles wherein all said lipids are non phospholipids, as well as their use as vehicle particularly in therapeutic applications such as prevention of AIDS. These non-phospholipid lipid vesicles comprise at least one external stabilized bilayer comprising amongst other a bilayer-modulating lipid chosen from the cholesterol family, an intravesicular aqueous space and at least one intravesicular micro-emulsion particle surrounded by an internal lipid monolayer. Inactivation of the HIV virus is due to the fusion of the non-phospholipid lipid vesicle with the membrane of said virus. This fusogenic property is probably due to the presence of cholesterol in the modulating lipid bilayer and there is no exchange of lipids between said non-phospholipid lipid vesicle containing cholesterol and the membrane of the HIV virus. Fusion between the nPLV described above and the membrane of an enveloped virus is not appropriate for in vivo inactivating said enveloped virus since it needs a long time to take place.
Despite the disclosure of the foregoing patents and patent applications, there remains therefore a need for a new method of inactivating an enveloped virus that is rapid and efficient, in vitro as well as in vivo.